Biology subjects

Chen, X.

Publications and source records attributed to Chen, X..

At least 397 records · Page 22Linked to original sources

bioRxiv2025

APOE genotype confers context dependent neurovascular vulnerability in immune-vascularized human forebrain organoids

The APOE gene is a major genetic determinant of neurovascular and immune function, yet the mechanisms by which its isoforms modulate brain vulnerability to pathogenic stress remain incompletely understood. Here, we employ isogenic human iPSC-derived immune-vascularized--Forebrain Organoid-based Multicellular Assembled Cerebral Organoids (FORMA-COs)--to dissect isoform-specific responses to a clinically relevant viral challenge. We find that APOE2/2 and APOE4/4 FORMA-COs exhibit heightened viral RNA burden and distinct neuroinflammatory profiles compared to APOE3/3. Specifically, APOE4/4 promotes IL-1 and VEGFA induction, whereas APOE2/2 leads to elevated TNF-{beta} and VEGFA protein accumulation, indicating divergent pathways of injury. Integrated transcriptomic analyses, combined with known and predicted APOE protein-protein interaction networks, reveal genotype-dependent enrichment of cytokine signaling, angiogenic remodeling, and immune dysregulation. In vivo validation using humanized mouse models corroborates APOE genotype- specific vascular remodeling, microglial activation, and oligodendrocyte perturbation. These findings demonstrate that APOE genotype confers context-specific susceptibility to neuroimmune and vascular injury, providing insight into genetic risk mechanisms underlying infection-related and neurodegenerative brain disorders.

neuroscience↗

bioRxiv2025

The Association Between SLIT2 in Human Vitreous Humor and Plasma and Neurocognitive Test Scores

BackgroundSlit Guidance Ligand 2 (SLIT2) binds Roundabout (ROBO) guidance receptors to direct axon pathfinding and neuron migration during nervous system development. SLIT2 expression has previously been linked to dementia risk. ObjectiveTo study the association between SLIT2 expression in human vitreous humor and plasma samples and neurocognitive test scores in a cross-sectional cohort study utilizing a novel, highly-sensitive Meso Scale Discovery (MSD) assay for SLIT2 detection. MethodsSeventy-nine individuals with a mean age of 55.79 {+/-} 12.03 years underwent eye surgery with collection of vitreous humor, blood (plasma) collection, and neurocognitive assessment. Vitreous humor and plasma samples were analyzed by SLIT2 MSD electrochemiluminescence immunoassay. Associations between SLIT2 levels in vitreous humor and plasma were analyzed using GraphPad Prism. ResultsWe found up to a 7-fold higher level of SLIT2 in human vitreous humor compared to plasma. Lower vitreous SLIT2 levels were associated with a lower Montreal Cognitive Assessment (MoCA) score and Immediate Recall Verbatim (IRV) z-score, and higher plasma SLIT2 was associated with a lower MoCA score. In multivariate analysis using single and multiple predictor models, the same significant associations were found when adjusted for age, sex, race, diabetic status, diabetic retinopathy status, glaucoma status, and Apolipoprotein E (APOE) genotype. ConclusionsSLIT2 protein levels are significantly associated with MoCA score and IRV z-score in middle-aged individuals. The relationship remained significant when adjusted for demographics, co-morbidity, and APOE genotype, suggesting SLIT2 may be a sensitive biomarker for detection of mild cognitive impairment and early dementia, and warrants further studies.

neuroscience↗

bioRxiv2025

MicroProphet: A Digital Twin Framework for Predicting Microbial Community Dynamics with Personalized Precision

The ability to accurately predict the dynamic evolution of microbial communities is critical for advancing personalized medicine, precision intervention, and ecological system management. However, the irregular sampling, high missingness, and complex temporal behaviors that characterize longitudinal microbiome datasets present substantial challenges to reliable forecasting. Here we propose MicroProphet, a personalized digital twin framework capable of accurately forecasting microbial abundance trajectories from incomplete longitudinal observations without the need for data interpolation. By leveraging a time-aware Transformer architecture, MicroProphet reconstructs individualized microbial trajectories using as little as the initial 30% of time points, capturing critical transitional states through its attention mechanism. We demonstrate its robust cross-ecosystem generalizability across synthetic communities, human gut microbiomes, infant gut development, and corpse decomposition. In clinical contexts, MicroProphet enables early identification of disease-related microbial shifts and supports intervention timing optimization, exemplified in inflammatory bowel disease and antibiotic perturbation responses. By transforming incomplete and sparse data into actionable forecasts, MicroProphet establishes a foundation for real-time microbial monitoring, therapeutic decision support, and precision ecological management, paving the way for broader applications of digital twin systems in biology and personalized healthcare. HighlightsO_LIMicroProphet establishes a personalized digital twin framework for forecasting biological dynamics from incomplete longitudinal observations, enabling precision health monitoring and intervention planning. C_LIO_LIBy leveraging a transformer-based architecture, MicroProphet accurately reconstructs microbial community trajectories using as little as 30% of initial time points, without the need for data interpolation. C_LIO_LIThe framework demonstrates robust cross-ecosystem generalizability across clinical, early-life development, and forensic contexts. C_LIO_LIMicroProphet empowers real-time tracking of microbial shifts, early detection of disease-associated transitions, and timing optimization for microbiome-targeted therapeutic strategies. C_LI

microbiology↗

bioRxiv2025

Glucocorticoid receptor and RUNX transcription factors cooperatively drive CD8 T cells dysfunction in human cancer

Glucocorticoids (GCs) are potent modulators of immune responses; however, the mechanisms by which GCs regulate gene expression in human CD8 T cells remain incompletely defined. Here, we delineate how physiological cortisol signalling shapes the transcriptional and chromatin landscapes of primary human CD8 T cells. We identify a substantial cohort of GC-responsive genes that are co-regulated through the cooperative activity of the glucocorticoid receptor (GR) and RUNX transcription factors. Integrative RNA sequencing and ChIP sequencing analyses identified genome-wide cortisol-responsive immunoregulatory genes. Genetic deletion of the GR (encoded by NR3C1) abolished cortisol-induced gene expression changes, confirming GR-dependency. Notably, GR chromatin occupancy in cortisol-treated CD8 T cells was strongly enriched at RUNX transcription factor (TF) motif rather than canonical GC response elements (GREs). Co-immunoprecipitation assays validated a ligand-dependent physical interaction between GR and RUNX and revealed the interacting domains. Single-cell transcriptomic analyses of tumour infiltrating CD8 T cells revealed significant enrichment of cortisol-responsive genes, indicating an active GC signalling (response) within the tumour microenvironment. GR-RUNX dual controlled genes were enriched in tumour-infiltrating CD8 T cells across multiple cancer types, including lung adenocarcinoma, head and neck squamous carcinoma, pancreatic cancer, and breast cancer. We found GR-RUNX co-regulated genes are predominantly expressed in the predysfunctional state of CD8 T cell population of different solid tumours. These results suggest that local cortisol signalling within tumour microenvironments drives CD8 T cell dysfunction through GR-RUNX TF cooperation. Collectively, our findings identify RUNX3 TF as a critical mediator of GR signalling in human CD8 T cells and reveal a novel mechanism by which endogenous GCs influence antitumour immunity, which could be therapeutically targetable.

immunology↗

bioRxiv2025

Macrophage Migration Inhibitory Factor Suppresses Natural Killer Cell Response and Promotes Hypoimmunogenic Stem Cell Engraftment Following Spinal Cord Injury

Human induced pluripotent stem cells (iPSCs) offer immense potential as a source for cell therapy in spinal cord injury (SCI) and other diseases. The development of hypoimmunogenic, universal cells that could be transplanted to any recipient without requiring a matching donor, could significantly enhance their therapeutic potential and accelerate clinical translation. To create off-the-shelf hypoimmunogenic cells, we used CRISPR-Cas9 to delete B2M (HLA class I) and CIITA (master regulator of HLA class II). Double-knockout (DKO) iPSC-derived neural progenitor cells (NPCs) evaded T cell-mediated immune rejection in vitro and after grafting into the injured spinal cord of athymic rats and humanized mice. However, loss of HLA class I heightened susceptibility to host natural killer (NK) cell attack, limiting graft survival. To counter this negative effect, we engineered DKO NPCs to overexpress macrophage migration inhibitory factor (MIF), an NK cell checkpoint ligand. MIF expression markedly reduced NK cell-mediated cytotoxicity and improved long-term engraftment and integration of NPCs in the animal models for spinal cord injury. These findings demonstrate that MIF overexpression, combined with concurrent B2M and CIITA deletion, generates hiPSC neural derivatives that escape both T- and NK-cell surveillance. This strategy provides a scalable route to universal donor cells for regenerative therapies in SCI and potentially other disorders.

cell biology↗

bioRxiv2025

ZapC crosslinks FtsZ filaments through a dual-binding mechanism modulated by the intrinsically disordered linker of FtsZ

Most bacteria divide through binary fission, which is mediated by a large protein complex called the divisome. Assembly of the divisome is initiated by formation a Z ring at midcell consisting of polymers of the bacterial tubulin FtsZ. A series of FtsZ-associated proteins (Zaps), which crosslink FtsZ filaments, promote Z ring formation in Escherichia coli. However, how these proteins interact with FtsZ is still unclear. In this study, we discover that ZapC binds to both FtsZs globular domain and its conserved C-terminal peptide (CCTP) to crosslink FtsZ filaments. An AlphaFold 3 structural model of the FtsZ-ZapC complex indicates that ZapC binds to the globular domain of FtsZ via a loop region connecting its N-terminal and C-terminal domains and to the CCTP of FtsZ via a hydrophobic pocket in the N-terminal domain. Substitutions in these regions of ZapC disrupt its binding to FtsZ, validating the dual binding mode. Strikingly, we find that the intrinsically disordered C-terminal linker (CTL) of FtsZ affects the interaction of FtsZ with ZapC as well as other partners, indicating an important role of the CTL in FtsZ functionality. Taken together, these results indicate that ZapC, although it exists as a monomer, can crosslink FtsZ filaments by a two-pronged mechanism, binding to the globular domain of FtsZ in one filament and to the CCTP of FtsZ in another filament. Furthermore, the CTL plays an important role in regulating FtsZ interaction with its partners. ImportanceBacterial cytokinesis requires the Z ring, a highly dynamic cytoskeletal element consisting of polymers of the bacterial tubulin FtsZ. Formation of a coherent and functional Z ring is facilitated by FtsZ-associated proteins (Zap), which can crosslink FtsZ polymers, but how these proteins work is still incompletely understood. In this study, we find that ZapC, one of the FtsZ crosslinkers, binds to both FtsZs globular domain and its conserved C-terminal peptide to crosslink FtsZ filaments. Moreover, the intrinsically disordered C-terminal linker (CTL) of FtsZ modulates its binding to ZapC and many other FtsZ binding proteins. These findings reveal a novel mechanism to crosslink FtsZ filaments and reveal an important and highly conserved role of the CTL in FtsZ functionality.

microbiology↗

bioRxiv2025

Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT-NOS Signaling

Nitrate functions as a signaling molecule beyond its metabolic intermediate role. Despite progress in plants, the mechanisms underlying mammalian nitrate sensing and signaling remain unclear. The accompanying study identifies Sialin2--a proteolytic fragment of nitrate transporter Sialin--as a mammalian nitrate sensor mediating cellular responses. Here, we demonstrate that nitrate triggers endocytosis, inducing Sialin proteolysis and Sialin2 generation. Nitrate-induced Sialin2 scaffolds Lyn kinase with epidermal growth factor receptor (EGFR) at endosomes, activating phosphatidylinositol 3-kinase (PI3K)-AKT-nitric oxide synthase (NOS) pathway to stimulate localized nitric oxide (NO) production, enhancing angiogenesis and cell survival. In hypertensive rats, nitrate supplementation restores endothelial function and reduces blood pressure through AKT/eNOS-dependent signaling. Unlike the classical nitrate-nitrite-NO pathway, the Sialin2-PI3K-AKT-NOS axis confines NO synthesis to endosomal microdomains, enabling spatiotemporally precise vasodilation. By establishing Sialin2 as a mammalian nitrate sensor, this study unveils a novel paradigm in nitrogen homeostasis and provides targeted therapeutic strategies for vascular disorders.

cell biology↗

bioRxiv2025

Sialin2 Functions as a Mammalian Nitrate Sensor to Sustain Mitochondrial Homeostasis

Nitrogen homeostasis is fundamental for cellular physiology, yet mammalian nitrate (NO3-) sensing mechanisms remain elusive. Here, we identify Sialin2--a proteolytic fragment of the nitrate transporter Sialin generated by cathepsin B (CTSB) cleavage-- as the first mammalian nitrate sensor. Microscale thermophoresis (MST) reveals Sialin2 as a high-affinity nitrate sensor, while Cryo-Electron Microscopy (Cryo-EM) uncovers its structural basis for signaling. We show that Sialin2 localizes to mitochondria and scaffolds liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) complexes to drive organelle-specific metabolic adaptation via spatiotemporally controlled AMPK activation, enhancing mitochondrial biogenesis, ATP production, and cell survival. Real-time tracking using the sCiSiNiS biosensor demonstrates nitrate signaling dynamics at physiological levels. This signaling axis redefines nitrate as a direct ligand activating receptor-like cascades, independent of classical nitric oxide synthesis. Our findings establish a paradigm of "inorganic salt signaling biology", wherein anions co-opt trafficking systems to achieve signaling specificity, offering therapeutic avenues for mitochondrial disorders.

cell biology↗

bioRxiv2025

Expression and Characterization of SARS-CoV-2 Spike Protein in Thermothelomyces heterothallica C1

The COVID-19 pandemic demonstrated a pressing need for rapid, adaptive, and scalable manufacturing of vaccines and reagents. With the transition into an endemic disease and rising threats of other emerging pandemics, production of these biologicals requires a stable and sustainable supply chain and accessible distribution methods. In this study, we demonstrate the strength of an engineered filamentous fungal platform, Thermothelomyces heterothallica C1, for high volumetric productivity of the full-length spike glycoprotein. Spike protein produced in this system is highly thermostable and immunization of mice with spike made in C1 or mammalian platforms resulted in a similar humoral response. Additionally, it was shown that the native N-glycan profile can be redecorated with complex sialylated structures, if necessary, resulting in a more human-like glycan profile, without impacting binding characteristics as shown experimentally and in simulations. Through extensive physicochemical analysis, the C1 produced spike performs similarly to spike proteins produced in other commercially available systems. The data presented is evidence that C1 can be a strong platform for production of complex glycosylated recombinant proteins such as subunit antigen vaccines.

bioengineering↗

bioRxiv2025

PIBE: Parallel ion beam etching of sections collected on wafer for ultra large-scale connectomics

We developed a parallel ion beam etching device that simultaneously thins hundreds of sections on a whole 4-inch wafer at 20 nm steps, enabling sub-centimeter volumetric imaging with high axial resolution. Compared with traditional volume electron microscopy, PIB preserves in-situ continuity within sections and simplifies inter-section registration. Its high axial resolution facilitates reliable neuron segmentation and tracing with reduced proofreading, providing an efficient route toward ultra-large-scale connectomics, such as whole-mouse-brain reconstruction.

neuroscience↗

bioRxiv2025

Breakthrough infection elicits synergistic broadly neutralizing antibodies targeting non-dominant epitopes on RBD and NTD in an individual of inactivated SARS-CoV-2 vaccine

Background/ObjectivesIdentification and characterization of broadly neutralizing monoclonal antibodies from individuals exposed to SARS-CoV-2, either by infection or vaccination, can inform the development of next-generation vaccines and antibody therapeutics with pan-SARS-CoV-2 protection. MethodsThrough single B cell sorting and RT-PCR, monoclonal antibodies (mAbs) were isolated from a donor who experienced BA.5 or BF.7 breakthrough infection after three doses of inactivated vaccines. Their binding and neutralizing capacities were respectively measured with ELISA and pseudovirus-based neutralization assay. Their epitopes were mapped by competition ELISA and site- directed mutation. ResultsAmong a total of 67 spike-specific mAbs cloned from the donor, four mAbs (KXD643, KXD652, KXD681, and KXD686) can neutralize all tested SARS-CoV-2 variants from wild-type to KP.3. Moreover, KXD643, KXD652, and KXD681 belong to a clonotype encoded by IGHV5-51 and IGKV1-13, and recognize the cryptic and conserved RBD-8 epitope on the receptor-binding domain (RBD). In contrast, KXD686 is encoded by IGHV1-69 and IGKV3-20, and targets a conserved epitope (NTD Site iv) outside the antigenic supersite (NTD Site i) of the N-terminal domain (NTD). Notably, antibody cocktails containing these two groups of mAbs can neutralize SARS-CoV-2 more potently due to synergistic effects. In addition, bispecific antibodies derived from KXD643 and KXD686 demonstrate further improved neutralizing potency compared to antibody cocktails. ConclusionsThese four mAbs can be developed as candidates of pan-SARS-CoV-2 antibody therapeutics through further antibody engineering. On the other hand, vaccines designed to simultaneously elicit neutralizing antibodies towards RBD-8 and NTD Site iv have the potential to provide pan-SARS-CoV-2 protection.

immunology↗

bioRxiv2025

Hierarchical small molecule inhibition of MYST acetyltransferases

MYST lysine acetyltransferases (KATs) is a class of epigenetic enzymes critical for cellular function that constitute an emerging therapeutic target in cancer. Recently, several drug-like MYST inhibitors have been reported that show promise in a variety of preclinical models as well as in clinical trials of breast cancer. However, the comparative properties of these small molecules remains to be directly assessed. Here we apply an integrated profiling strategy to systematically define the potency and selectivity of drug-like MYST KAT inhibitors. First, we use optimized chemoproteomic profiling and histone acetylation biormarkers to study the industry-developed KAT inhibitor PF-9363. This reveals dose-dependent engagement of native KAT complexes, with hierarchical inhibition following the order KAT6A/B > KAT7 >> KAT8 > KAT5. Next, we demonstrate how PF-9363s ability to disrupt capture of MYST complex members in chemoproteomic experiments can be leveraged to identify new candidate members of these complexes, including the transcription factor FOXK2. Applying insights from these studies to WM-8014, WM-1119 and WM-3835, which have been extensively applied in the literature as MYST probes, highlights unexpected cross-inhibition and suggests a new framework for how these small molecules and biomarkers may be applied to differentiate KAT6A/B and KAT7-dependent phenotypes. Finally, we benchmark the activity of PF-9363 in the NCI-60 cell line screen, providing evidence that it can inhibit the growth of cell lines that are resistant to other epigenetic inhibitors by engaging the essential MYST enzyme KAT8 at high concentrations. Collectively, our studies indicate the potential for MYST KAT inhibitors to exhibit dose-dependent target engagement reminiscent of kinase inhibitors and specify assays and biomarkers for facile monitoring of selective and hierarchical effects.

biochemistry↗

bioRxiv2025

Cell type- and chromosome-specific chromatin landscapes and DNA replication programs of Drosophila testicular stem cells

Stem cells have the unique ability to self-renew and differentiate into specialized cell types. Epigenetic mechanisms, including histones and their post-translational modifications, play a crucial role in regulating programs integral to a cells identity, like gene expression and DNA replication. However, the transcriptional, chromatin, and replication timing profiles of adult stem cells in vivo remain poorly understood. Containing germline stem cells (GSCs) and somatic cyst stem cells (CySCs), the Drosophila testis provides an excellent in vivo model for studying adult stem cells. However, the small number of stem cells and cellular heterogeneity of this tissue have limited comprehensive genomic studies. In this study, we developed cell type-specific genomic techniques to analyze the transcriptome, histone modification patterns, and replication timing of GSC-like and CySC-like cells. Single cell RNA sequencing validated previous findings on GSC-CySC intercellular communication and revealed high expression of chromatin regulators in GSC-like cells. To characterize chromatin landscapes, we developed a cell-type-specific chromatin profiling assay to map H3K4me3-, H3K27me3-, and H3K9me3- enriched regions, corresponding to euchromatic, facultative heterochromatic, and constitutive heterochromatic domains, respectively. Finally, we determined cell type-specific replication timing profiles, integrating our in vivo datasets with published data using cultured cell lines. Our results reveal that GSC-like cells display a distinct replication program compared to somatic lineages, that aligns with chromatin state differences. Collectively, our integrated transcriptomic, chromatin, and replication datasets provide a comprehensive framework for understanding genome regulation differences between these in vivo stem cell populations, demonstrating the power of multi-omics in uncovering cell type- specific regulatory features.

genomics↗

bioRxiv2025

Exploring non-invasive sexing of early chick embryos in intact eggs using Laser Speckle Contrast Imaging (LSCI) and Deep Neural Network (DNN)

The ability to image blood flow in early-stage avian embryos has significant applications in developmental biology, drug and vaccine testing, as well as determining sex differentiation. In this project, we used our recently developed laser speckle contrast imaging (LSCI) system to non-invasively image extraembryonic blood vessels and used these images to attempt early sex identification of chick embryos. Specifically, we captured images of blood vessels from 1,251 living chicken embryos between day three and day four of incubation. We then applied deep neural network (DNN) models to evaluate whether it is possible to differentiate sex based on vascular patterns. Using ResNetBiT and YOLOv5 models, our results indicate that sex differentiation from extraembryonic blood vessel images was not achievable with sufficiently high accuracy or statistical significance for practical use. Specifically, ResNetBiT had a five-fold cross-validated average accuracy of 59%{+/-}5% (fold-wise p-value, p [≤] 0.3) at day 3 and 61%{+/-}3% (fold-wise, p [≤] 0.04) at day 4. YOLOv5 had a five-fold cross-validated average accuracy of 55%{+/-}3% (fold-wise, p [≤] 0.3) at day 3 and 53%{+/-}3% (fold-wise, p [≤] 0.5) at day 4. Our findings suggest that using vascular pattern imaging alone is inconclusive for reliable early sex identification in chicken embryos.

bioengineering↗

bioRxiv2025

Gut Microbiota Dysbiosis and Short-chain Fatty Acid alterations in Pediatric New-Onset Type 1 diabetes with Ketoacidosis

ObjectiveDiabetic ketoacidosis (DKA) stands as the most common acute hyperglycaemic complication with a relatively high mortality rate among children diagnosed with type 1 diabetes (T1D). Previous studies have shed light on gut dysbiosis in newly-onset T1D children, but research targeting the gut microbiota and microbial metabolites in patients with DKA remains scarce. MethodsShotgun metagenomic analysis was conducted on fecal samples from 96 newly diagnosed T1D children, including 32 patients who presented with DKA upon admission. Short-chain fatty acids (SCFAs) were quantified using gas chromatography/mass spectrometry (GC/MS). Comparative analyses were performed on the gut microbiome and SCFA levels between DKA and non-DKA subgroups. ResultsGut microbiota composition differed between DKA and non-DKA groups. While alpha-diversity remained consistent, beta-diversity showed marked differences between the two groups, and the phyla Firmicutes and Bacteroidetes exhibited predominance in the DKA group. DKA was associated with increased potential pathogens and a significant depletion of SCFA-producing genera (Anaerobutyricum, Dialister, Ruminococcus, Roseburia, Dorea, and Butyricicoccus), accompanied by reduced SCFA levels. Further correlation analysis revealed strong associations between SCFAs, SCFA-producing microbiota, and disease severity indicators. Propionic acid and butyric acid mediated the association between SCFA-producing bacteria and DKA. ConclusionThis study first reported reductions in SCFAs and SCFA-producing bacteria in the fecal samples of T1D children at DKA onset. Our findings highlight SCFAs and their producers as protective biomarkers of DKA and suggest that targeting gut microbiota and SCFA metabolism may offer a potential therapeutic strategy for DKA. ImportanceWhile gut microbiota imbalances have been linked to type 1 diabetes (T1D), little is known about the role of gut microbes and their metabolites during acute diabetic ketoacidosis (DKA), an acute and life-threatening complication. This study examined the gut microbiome and levels of short-chain fatty acids (SCFAs) in fecal samples from children with new-onset T1D with and without DKA. DKA was characterized by distinct microbial signatures, including increased potential pathogens, a loss of beneficial SCFA-producing bacteria, and consistently lower SCFA levels. These changes closely correlated with indicators of disease severity, and further mediation analysis revealed the role of SCFAs in linking gut microbiota to DKA. These findings suggest that SCFAs and their producers may serve as protective markers for DKA, highlighting the potential of microbiome-based interventions to improve outcomes in children facing this critical complication.

microbiology↗

bioRxiv2025

AAVs Targeting Human Carbonic Anhydrase IV Enhance Gene Delivery to the Brain

Clinically approved gene therapies based on natural adeno-associated virus (AAV) serotypes have restricted applications, particularly in the brain, due to their poor targeting, high dose requirements, and resulting safety concerns. Directed evolution of enhanced AAV capsids in mice or non-human primates (NHPs) has resulted in markedly improved performance in those species, but inter-species differences present a serious challenge for translating these vectors into human therapies. Here, we engineer AAVs to target human carbonic anhydrase IV (CA-IV), a recently identified blood-brain barrier (BBB) transcytosis receptor. Among known transcytosis receptors, CA-IV is notable for its relatively specific expression in brain endothelial cells and the potency of AAVs that target it in mice. CA-IVs AAV binding site, and thus the mouse vectors enhanced brain potency, is not conserved across species, so we employed a two-phase engineering strategy to identify AAVs optimized for human CA-IV-dependent gene delivery to the brain. We first used in vitro receptor-based selection of a vast AAV library to exclude capsids that do not bind human CA-IV, followed by in vivo selection in "humanized" mice expressing human CA-IV in brain endothelial cells. Notably, we find that human CA-IV binding capsid variants that were poorly enriched in the pull-down selection outperform strong binders in vivo. The most promising vector, AAV-hCA4-IV77, engages human CA-IV to achieve 100-fold greater brain transduction than AAV9, with robust neuronal and astrocytic coverage throughout multiple brain regions. These results advance our understanding of receptor-targeted capsid design and support the therapeutic potential of human CA-IV-engaging AAVs.

bioengineering↗

bioRxiv2025

Finite element model predicts micromotion-induced strain profiles that correlate with the functional performance of Utah arrays in humans and non-human primates

ObjectiveUtah arrays are widely used in both humans and non-human primates (NHPs) for intracortical brain-computer interfaces (BCIs), primarily for detecting electrical signals from cortical tissue to decode motor commands. Recently, these arrays have also been applied to deliver electrical stimulation aimed at restoring sensory functions. A key challenge limiting their longevity is the micromotion between the array and cortical tissue, which may induce mechanical strain in surrounding tissue and contribute to performance decline. This strain, due to mechanical mismatch, can exacerbate glial scarring around the implant, reducing the efficacy of Utah arrays in recording neuronal activity and delivering electrical stimulation. ApproachTo investigate this, we employed a finite element model (FEM) to predict tissue strains resulting from micromotion. Main ResultsOur findings indicated that strain profiles around edge and corner electrodes were greater than those around interior shanks, affecting both maximum and average strains within 50 {micro}m of the electrode tip. We then correlated these predicted tissue strains with in-vivo electrode performance metrics. We found negative correlations between 1 kHz impedance and tissue strains in human motor arrays and NHP area V4 arrays at 1-mo, 1-yr, and 2-yrs post-implantation. In human motor arrays, the peak-to-peak waveform voltage (PTPV) and signal-to-noise ratio (SNR) of spontaneous activity were also negatively correlated with strain. Conversely, we observed a positive correlation between the evoked SNR of multi-unit activity and strain in NHP area V4 arrays. SignificanceThis study establishes a spatial dependence of electrode performance in Utah arrays that correlates with tissue strain.

neuroscience↗

bioRxiv2025

Design and Biological Activity of a Novel Brain Penetrant Urea Compound Against Glioblastoma

Glioblastoma (GBM) remains the most lethal primary brain tumor, largely due to therapy-resistant glioma stem cells (GSCs) and the ability of non-stem cells to dedifferentiate under therapeutic pressure. We developed MXC-017, a novel urea-based compound that crosses the blood-brain-barrier, directly targets GSCs, and prevents radiation-induced GSC formation. Using click chemistry pull-down and mass spectrometry, we identified vimentin as the target of MXC-017, further validated by in silico docking. Global transcriptomic profiling (bulk RNA-seq) and single-cell RNA-seq analyses revealed MXC-017s efficacy with minimal off-target effects, supported by metabolic and kinome assays. Normal cell toxicity was negligible in fibroblasts, microglia, astrocytes, and murine neural progenitors. Maximum tolerated dose was identified and we observed significantly extended median survival in 17 PDOX GBM models when treated with MXC-017 plus radiation, benchmarked against standard-of-care temozolomide. These findings underscore the therapeutic potential of vimentin-targeting agents to overcome radiation resistance and improve outcomes for GBM patients. Statement of SignificanceGlioblastomas distinctive nature and the blood-brain barrier hamper therapies targeting therapy-resistant GSCs. We developed a novel urea-based agent that crosses the barrier, targets GSCs, and prevents radiation-induced GSC formation. With minimal off-target effects, reduced toxicity, and superior survival in PDOX models, it offers potential to improve outcome in GBM.

neuroscience↗